Aminoglycoside antibiotics are essential for battling life-threatening bacterial sepsis. Aminoglycosides also cause permanent deafness/balance disorders and nephrotoxicity in more than 120,000 individuals each year in the US, particularly in infants and premature babies. The long-term goal of this research is to prevent cochlear uptake of aminoglycosides and thus ototoxicity, to preserve auditory function. Progress in the previous project period revealed that the cellular uptake of aminoglycosides can be blocked in vitro, and that non-selective cation channels enhance the clearance of aminoglycosides. In this proposal, we will investigate how aminoglycosides are transported from the vasculature, across the blood-labyrinth barrier into the cochlear fluids and tissues in vivo, and develop strategies to this transport to prevent aminoglycoside-induced inner ear sensory hair cell death. Our working hypothesis is that: pharmacological agents can reduce aminoglycoside uptake and toxicity in the cochlea. The specific aims of this project are: First, to regulate aminoglycoside uptake in vitro, by identifying how pharmacological agents change the intra-cellular milieu of model cell lines and cochlear explants to enhance or inhibit aminoglycoside uptake and clearance. We will monitor intracellular calcium and pH levels, the resting potential and membrane resistance (Aim 1). Second, to preserve auditory function and morphology in vivo using inhibitors of aminoglycoside uptake. We will assess the efficacy of aminoglycoside-uptake inhibitors using auditory brainstem response audiometry, confocal microscopy of hair cell morphology, and constructing cytocochleograms (Aim 2). And thirdly, to identify the intra-cochlear route of aminoglycosides from the vasculature to the sensory hair cells. We will use cochlear perfusion techniques, and sample cochlear fluids to determine if aminoglycosides enter hair cells from endolymph or perilymph. We will then verify that aminoglycoside uptake-inhibitors reduce the intra-cochlear transport of aminoglycosides by one or both routes (Aim 3). Identification of mechanisms that reduce aminoglycoside transport into, and within, the cochlea will determine if currently-available drugs can be used as co-therapeutics to preserve auditory function during life-saving aminoglycoside therapy. In addition, identifying intracellular mechanisms (drug-induced or otherwise) that enhance aminoglycoside uptake provides new insight for clinicians to screen patients for pre-existing conditions and medications that elevate the risk of aminoglycoside toxicity in patients. Understanding mechanisms of how aminoglycosides antibiotics cross the blood-labyrinth barrier to enter the inner ear fluids is crucial in preventing aminoglycoside-induced ototoxicity. The proposed research will enable us to develop strategies to prevent the entry of aminoglycosides into the inner ear fluids and therefore ototoxic sequelae, particularly life-long deafness, tinnitus and vestibular deficits.